In vitro activity: Encorafenib (formerly known as LGX818) is a new-generation, highly potent, and orally bioavailable B-RAF(V600E) inhibitor with IC50 of 4 nM. It has little effect against wild-type BRAF. Encorafenib has selective anti-proliferative and apoptotic activity on cells expressing BRAFV600E. It shows no significant activity against a panel of 100 kinases and no suppression of cell growth with more than 400 cell lines expressing BRAFV600E. In human melanoma xenograft models, oral administration of Encorafenib shows strong decrease in phospho-MEK and induces tumor regression. Raf kinase is a serine/threonine enzyme in the RAFMAPK/ERK signaling pathway. By inhibiting the activation of the RAF/MAPK/ERK signaling pathway, encorafenib may result in a decrease in proliferation of tumor cells. In the A375 (BRAFV600E) human melanoma cell line LGX818 suppresses phospho-ERK (EC50 = 3 nM) leading to potent inhibition of proliferation (EC50 = 4 nM). No significant activity is observed against a panel of 100 kinases (IC50> 900 nM) and LGX818 does not inhibit proliferation of> 400 cell lines expressing wild-type BRAF. Contributing to the high potency of LGX818 is the extremely slow off-rate from BRAFV600E which is not observed with other RAF inhibitors. In biochemical assays the dissociation half-life is>24 hours which translated into sustained target inhibition in cells following drug wash-out.

Kinase Assay: The Raf kinase activity reaction is started by the addition of 10 μL per well of 2×ATP diluted in assay buffer. After 3 hours (bRaf(V600E)) or 1 hour (c-Raf), the reactions are stopped with the addition of 10 μL of stop reagent (60 mM EDTA). Phosphorylated product is measured using a rabbit anti-p-MEK antibody and the Alpha Screen IgG (ProteinA) detection Kit, by the addition of 30 μL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in bead buffer (50 mM Tris, pH 7.5, 0.01% Tween20). The additions are carried out under dark conditions to protect the detection beads from light. A lid is placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence is read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) is calculated by non-linear regression using XL Fit data analysis software.

Cell Assay: A375 is a melanoma cell line that harbors the B-Raf V600E mutation. A375-luc cells engineered to express luciferase is plated to 384-well white clear bottom plates as 1,500 cells/50 μL/well in DMEM containing 10% FBS. Test compounds, dissolved in 100% DMSO at appropriate concentrations, are transferred to the cells by a robotic Pin Tool (100 mL). The cells are incubated for 2 days at 25°C, then 25 μL of BrightGloTM is added to each well and the plates are read by luminescence. The concentration of each compound for 50% inhibition (IC50) is calculated by non-linear regression using XL Fit data analysis software. wild type and V600E B-Raf.